Lysophosphatidic acid detection

ABSTRACT

Embodiments of methods and compounds for isolating and detecting lysophosphatidic acids (LPAs) are disclosed. Kits for performing the methods also are disclosed. LPAs are isolated from biological samples by liquid-liquid extraction followed by solid phase extraction. LPA species may be separated by HPLC, and the separated species may be identified and quantified. Also disclosed are embodiments of compounds capable of universally detecting a plurality of LPA species with substantially equivalent sensitivity. Embodiments of the disclosed compounds are useful for determination of total LPA concentration in a sample comprising a plurality of LPA species without separation of individual LPA species.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a reissue of U.S. Pat. No. 9,952,231, issued Apr. 24, 2018,which is the U.S. National Stage of International Application No.PCT/US2014/029025, filed Mar. 14, 2014, which was published in Englishunder PCT Article 21(2), which in turn claims the benefit of U.S.Provisional Application No. 61/790,443, filed Mar. 15, 2013, each ofwhich is incorporated herein in its entirety by reference.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant No. RO1CA136491 awarded by the National Institutes of Health, and Grant No.0741993, awarded by the National Science Foundation. The government hascertain rights in the invention.

FIELD

This disclosure concerns embodiments of methods and compounds forisolating and detecting lysophosphatidic acids. Kits for performing themethods also are disclosed.

BACKGROUND

Lysophosphatidic acid (LPA) is a bioactive phospholipid that stimulatescell proliferation, migration, and survival. Five G-protein-coupledreceptors have been identified as specific for LPA. Aberrant LPAproduction, expression, and signalling have been linked to cancerinitiation (e.g., tumorigenesis), progression, angiogenesis, andmetastasis. LPA is thought to play a role in a number of cancers,including ovarian cancer and other gynecological cancers. LPA also hasbeen linked to cardiovascular disease (e.g., atherosclerosis,atherothrombosis), platelet aggregation, ischemia perfusion injury,wound healing, neuropathic pain, neuropsychiatric disorders,reproductive disorders, and fibrosis.

In recent years, LPA has been considered as an important and sensitivebiomarker of ovarian cancer. Elevated LPA levels in plasma have beenfound in patients with ovarian cancer. There is evidence suggesting thatonly certain LPA species (below) are associated with ovarian cancer(Sutphen et al., Cancer Epidemiol. Biomark. Prev., 2004, 13:1185-1191;Xu et al. JAMA, 1998, 280:719-732), therefore, quantification ofindividual LPA species would provide a better way to improve theaccuracy of diagnosis. A need also exists for a colorimetric orfluorometric probe capable of detecting equal concentrations ofindividual LPAs with the same degree of response to more accuratelydetermine total LPA concentration with a single probe.

Known methods suffer from disadvantages. Xu et al. (JAMA, 1998,280:719-723) used a gas chromatography (GC) method to quantify total LPAplasma levels. Chen et al. (J. Chromatogr. B. Biomed. Sci. Appl., 2001,753:355-363) used capillary electrophoresis (CE) to quantify individualLPAs with an indirect ultraviolet (UV) for the detection. However, toseparate LPA from other lipids before the detection, these and severalother studies rely on two-dimensional thin layer chromatography (TLC) asa step, which is time consuming and labor intensive.

Holland et al. (J. Lipid Res., 2003, 44:854-858) used high performanceliquid chromatography (HPLC) to separate LPA species and evaporativelight-scattering detection (ELSD). This method avoids thetwo-dimensional TLC step, but LPA elutes at 38 min with a relatively lowrecovery. LC-MS based methodology has been used to quantify LPAs (Bakeret al., Anal. Biochem., 2001, 292:287-295); however, it is not asaccurate as LC-MS/MS because LC-MS only determines LPA by detecting themolecular mass ion rather than the parent to daughter ion transitions.

According to some recent studies, LC-MS/MS methods have disadvantages.Shan et al. (J. Chomatogr. B, 2008, 864:22-28) found that some unknowncompounds in plasma, which produced the same parent-to-daughter iontransition as LPA in a direct flow injection LC-ESI-MS/MS method, couldreduce the accuracy of quantification of LPA. Zhao et al. (J. LipidRes., 2010, 51:652-659) reported lysophosphatidylcholine (LPC) andlysophosphatidylserine (LPS) could lose the choline or serine group togenerate LPA-like signals at the ion source. Additionally, phosphatidicacids may be fragmented by enzymes during separation from blood samplesand/or fragmented in an ESI detector, in some instances losing one lipidchain and producing a false positive by appearing as LPA. Anotherdisadvantage is that LPAs do not ionize well, and the best results forLPA typically are obtained by running the mass spectrometer in negativeion mode, which can be more technically challenging than the moretypical positive ion mode.

SUMMARY

Embodiments of methods and compounds for isolating and detectinglysophosphatidic acids are disclosed. Kits for performing the methodsalso are disclosed.

Embodiments of compounds suitable for detecting lysophosphatidic acidshave a structure according to any one of general formulas I-IV.

With respect to general formula I, R¹-R⁶ independently are H, hydroxyl,thiol, lower alkyl, carboxyalkyl, amino, lower alkoxy, or halogen;R⁷-R¹⁰ independently are H, alkyl, acyl, carboxyl, nitro, amino,alkylamino, or —SO₃H; R¹¹ is N—C(═R¹³)—NH₂, N—NH—C(═R¹³)—NH₂,N—C(NH₂)═N—C(═R¹³)—NH₂, or N—NH—C(NH₂)═N—C(═R¹³)—NH₂, where R¹³ is O, S,or NH; each R¹² independently is hydrogen or lower alkyl, or each of R¹,R², R⁵, and R⁶ may together with an adjacent R¹² and N atom form a6-membered heterocyclic ring; and X is O, S, CH₂, NH, or SiR¹⁴ where R¹⁴is H or lower alkyl.

With respect to general formula II, R¹-R⁵, R¹⁵, R¹⁷, and R¹⁸independently are hydrogen, hydroxyl, thiol, lower alkyl, carboxyalkyl,amino, lower alkoxy, or halogen; R¹⁶ is —N(R¹²)₂; each R¹² independentlyis hydrogen or lower alkyl, or each of R¹, R², R¹⁵, and R¹⁷ may togetherwith an adjacent R¹² and N atom form a 6-membered heterocyclic ring; andR⁷-R¹⁰, R¹¹ and X are as described for general formula I.

With respect to general formula III, R²-R⁵, R¹⁵, R¹⁷, and R¹⁹-R²¹independently are hydrogen, hydroxyl, thiol, lower alkyl, carboxyalkyl,amino, lower alkoxy, or halogen; one of R¹⁶ and R¹⁸ is hydrogen,hydroxyl, thiol, lower alkyl, carboxyalkyl, amino, lower alkoxy, orhalogen, and the other of R¹⁶ and R¹⁸ is —N(R¹²)₂; each R¹²independently is hydrogen or lower alkyl, or if R¹⁶ is —N(R¹²)₂, each ofR¹⁵, R¹⁷, R²⁰, and R²¹ may together with an adjacent R¹² and N form a6-membered heterocyclic ring; and R⁷-R¹⁰, R¹¹ and X are as described forgeneral formula I.

With respect to general formula IV, R¹-R⁴, R⁶, and R²²-R²⁴ independentlyare hydrogen, hydroxyl, thiol, lower alkyl, carboxyalkyl, amino, loweralkoxy, or halogen; each R¹² independently is hydrogen or lower alkyl,or each of R¹, R², R²³, and R²⁴ may together with an adjacent R¹² and Nform a 6-membered heterocyclic ring; and R⁷-R¹⁰, R¹¹ and X are asdescribed for general formula I.

Exemplary compounds include, but are not limited to

Some embodiments of a method for isolating lysophosphatidic acid speciesfrom a plasma or serum sample include obtaining a plasma or serumsample; combining one part plasma or serum sample with five partssolvent comprising methanol and chloroform in a ratio of 2:1 to form amixture; incubating the mixture at 4° C. for 15-30 minutes; warming themixture to ambient temperature; separating organic and aqueous layers ofthe mixture; extracting the aqueous layer with phosphate-bufferedsaline, pH 7.4 to form an extracted aqueous phase; mixing the extractedaqueous phase with chloroform; separating chloroform from the extractedaqueous phase to form a washed aqueous phase; repeating the steps ofmixing the extracted phase with chloroform and separating chloroformfrom the extracted aqueous phase to form a washed aqueous phase; addingphosphoric acid to the washed aqueous phase to reduce pH to 2, therebyforming an acidified aqueous phase; loading the acidified aqueous phaseonto a solid-phase extraction (SPE) cartridge, wherein the SPE cartridgehas a C8 stationary phase; flowing water and subsequently chloroformthrough the SPE cartridge; drying the SPE cartridge; and flowingmethanol through the SPE cartridge, thereby eluting lysophosphatidicacid species in methanol from the SPE cartridge. In some embodiments,the method further includes evaporating methanol to form a dry residuecomprising lysophosphatidic acid species; and dissolving the dry residuein 9:1 methanol:H₂O to produce an extracted lysophosphatidic acid samplecomprising one or more lysophosphatidic acid species.

In some embodiments, a total concentration of lysophatidic acid (LPA)species in the extracted lysophosphatidic acid sample is determined. Thesample may be obtained from a subject suspected of being at risk of acondition associated with an aberrant LPA level, the method furthercomprising determining a risk level for the condition, wherein the risklevel is based at least in part on the lysophosphatidic acidconcentration. In certain embodiments, the condition is cancer (forexample, ovarian cancer), cardiovascular disease, platelet aggregation,ischemia perfusion injury, neuropathic pain, a neuropsychiatricdisorder, a reproductive disorder, or fibrosis. Total concentration ofLPA can be determined by combining the extracted lysophosphatidic acidsample with a compound according to any one of general formula I-IV in asolvent comprising 2.5-10% dimethylsulfoxide in methanol to form asolution; exposing the solution to a light source; measuringfluorescence intensity of the solution; and determining, based on thefluorescence intensity, the total concentration of lysophosphatidic acidspecies. In certain examples, the compound is

and fluorescence intensity is measured at 570 nm.

In some embodiments, lysophosphatidic acid species in the extractedlysophosphatidic acid sample are separated using a reversed-phasehigh-performance liquid chromatography (HPLC) column, and individuallysophosphatidic acid species are detected as the separatedlysophosphatidic acid species exit the reversed-phase HPLC column. LPAspecies may be separated using HPLC by flowing the sample into areversed-phase HPLC column including a C8 stationary phase, andsubsequently flowing 16:5 methanol/phosphate buffer (50 mM, pH 2.5)through the reversed-phase HPLC column, thereby forming an eluentcomprising lysophosphatidic acid species. Individual LPA species may bedetected by combining the eluent with4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide (DiA) as itexits the reversed-phase HPLC column to form a DiA-eluent mixture,flowing the DiA-eluent mixture through a detector, and detectingindividual lysophosphatidic acid species by detecting fluorescence ofthe DiA-eluent mixture as the DiA-eluent mixture flows through thedetector.

In certain embodiments, the method further includes identifying anindividual lysophosphatidic acid species by comparing an elution timefor the individual lysophosphatidic acid species to elute from thereversed-phase HPLC column to elution times for known individuallysophosphatidic acid species, measuring fluorescence intensity of theDiA-eluent mixture, and determining, based on the fluorescenceintensity, a concentration of the individual lysophosphatidic acidspecies. In some examples, the sample is obtained from a subjectsuspected of being at risk of a condition associated with an aberrantLPA level, and the method includes determining a risk level for thecondition, wherein the risk level is based at least in part on anidentification of an individual lysophosphatidic acid species, theconcentration of the individual lysophosphatidic acid species, or acombination thereof. Exemplary conditions that may be associated with anaberrant LPA level include cancer, cardiovascular disease, plateletaggregation, ischemia perfusion injury, neuropathic pain, aneuropsychiatric disorder, a reproductive disorder, or fibrosis. Incertain examples, the condition is ovarian cancer.

Embodiments of kits for detecting and quantifying lysophosphatidic acidinclude at least one compound according to any one of general formulasI-IV. The kits may further include one or more lysophosphatidic acidspecies, and/or one or more solid-phase extraction cartridges whereinthe stationary phase is C8.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows absorbance spectra of an aqueous solution of 3 μM4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide (DiA) in theabsence (dashed line) or presence (solid line) of 10 μM lysophosphatidicacid (LPA) 18:0.

FIG. 2 shows fluorescence emission spectra of 3 μM DiA in the absence(dashed line) or presence (solid line) of 10 μM LPA 18:0(λ_(ex/em)=470/590 nm).

FIG. 3 shows emission spectra and a calibration curve (inset) of 2.67 μMDiA upon titration with LPA (18:0).

FIG. 4 is an HPLC trace of a LPA mixture (10 μM LPA 14:0, 16:0, 18:0,18:1, 20:4 and 20 μM LPA 17:0) injected in a 20 μL injection loop.Chromatography conditions: Column: LUNA™ C8 column, 3 μm, 50×2.1 mm,mobile phase: methanol:phosphate buffer (50 mM, pH 2.5) 16:5; flow rate:0.32 mL/min. Injection volume: 20 μL. Sample concentration: 10 μM inmethanol:H₂O 9:1. Post-column reagent: 10 μM DiA in H₂O. Reagent flowrate: 0.62 mL/min. Detection wavelength: ex/em 450/570 nm.

FIGS. 5A-5E are a series of calibration curves of LPAs: 14:0—FIG. 5A,16:0—FIG. 5B, 18:0—FIG. 5C, 18:1—FIG. 5D, and 20:4—FIG. 5E. The curveswere obtained using the disclosed HPLC post-column procedure. The arearatio (A/As) is the peak area of the individual LPA divided by the peakarea of the internal standard (LPA 17.0).

FIGS. 6A-6E are a series of calibration curves LPAs: 14:0—FIG. 6A,16:0—FIG. 6B, 18:0—FIG. 6C, 18:1—FIG. 6D, and 20:4—FIG. 6E. The curveswere obtained using an LC/ESI/MS/MS method. The area ratio (A/As) is thepeak area of the individual LPA divided by the peak area of the internalstandard (LPA 17.0).

FIG. 7 is an HPLC trace of 10 μM LPAs mixture (LPA 14:0, 16:0, 17:0,18:0, 18:1 and 20:4) and LPAs isolated from Donor A human plasma.Chromatography conditions: Column—reversed phase C8, 3 μm, 50×2.0 mm;mobile phase—methanol:phosphate buffer (pH 2.5)=16:5; flow rate—0.32mL/min; injection volume—20 μL; sample concentration—10 μM in MeOH:H₂O9:1; post-column reagent—10 μM DiA in H₂O; reagent flow rate—0.62mL/min. Detection wavelengths: ex/em 450/570 nm.

FIGS. 8A and 8B are LC/ESI/MS/MS traces of a 10 μM mixture of standardLPAs (FIG. 8A) and a plasma sample from donor A (FIG. 8B). Column: LUNA™C8 column (50×2 mm, 3 μm) at 40° C. Injection volume; 10 μL. Mobilephase; MeOH:aqueous formic acid (pH 2.5) 9:1. Flow rate of 0.4 mL/min.Parent and daughter ions were detected in the negative ion mode, sprayervoltage; 3.0 kV, capillary temperature at 300° C.

FIG. 9 is a bar graph illustrating the relative fluorescence emissionobtained from two embodiments of the disclosed probes (GRBI and GRBII)when combined with LPA 16:0; Ex/Em=550 nm/570 nm, final probeconcentration 5 μM, final LPA16:0 concentration 10 μM, solvent systemDMSO 2.5% in chloroform.

FIG. 10 shows fluorescence spectra of GRBII alone and GRBII in thepresence of LPA14:0, 16:0, 18:0, 18:1, ex/em=550 nm/570 nm, final probeconcentration 5 μM, final LPA concentration 10 μM, solvent system CHCl₃:DMSO 9:1.

FIG. 11 is a graph of fluorescence intensity of GRBII alone and in thepresence of LPA14:0, 16:0, 18:0, 18:1 over time, ex/em=550 nm/570 nm,final probe concentration 5 μM, final LPA concentration 10 μM, solventsystem CHCl₃: DMSO 9:1.

DETAILED DESCRIPTION

Embodiments of methods and compounds for isolating and detectinglysophosphatidic acids (LPAs) are disclosed, along with kits forperforming the method. Embodiments of the method include a liquid-liquidand solid-phase extraction process to isolate LPAs in plasma frominterfering components. Following extraction, individual LPA species maybe separated by HPLC and detected in a fluorometric assay.Alternatively, total LPA may be quantified using embodiments of afluorescent compound capable of detecting a plurality of LPA specieswith substantially similar sensitivity.

I. TERMS AND ABBREVIATIONS

The following explanations of terms and abbreviations are provided tobetter describe the present disclosure and to guide those of ordinaryskill in the art in the practice of the present disclosure. As usedherein, “comprising” means “including” and the singular forms “a” or“an” or “the” include plural references unless the context clearlydictates otherwise. The term “or” refers to a single element of statedalternative elements or a combination of two or more elements, unlessthe context clearly indicates otherwise.

Unless explained otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this disclosure belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. The materials, methods, and examples areillustrative only and not intended to be limiting. Other features of thedisclosure are apparent from the following detailed description and theclaims.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, percentages, and soforth, as used in the specification or claims are to be understood asbeing modified by the term “about.” Accordingly, unless otherwiseindicated, implicitly or explicitly, the numerical parameters set forthare approximations that may depend on the desired properties sought,limits of detection under standard test conditions/methods, limitationsof the processing method, and/or the nature of the parameter orproperty. When directly and explicitly distinguishing embodiments fromdiscussed prior art, the embodiment numbers are not approximates unlessthe word “about” is recited.

Acyl: An organic functional group having the general formula —C(O)R,where R is alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic,aryl, or heteroaryl.

Alkoxy: A radical (or substituent) having the structure —O—R, where R isa substituted or unsubstituted alkyl. Methoxy (—OCH₃) is an exemplaryalkoxy group.

Alkyl: A hydrocarbon group having a saturated carbon chain. The chainmay be cyclic, branched or unbranched. The term lower alkyl means thechain includes 1-10 carbon atoms.

Alkylamino: An alkyl group where at least one hydrogen is substitutedwith an amino, mono-substituted amino or di-substituted amino group.

Amino: A chemical functional group —N(R)R′ where R and R′ areindependently hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic,heteroaliphatic, aryl (such as optionally substituted phenyl or benzyl),heteroaryl, alkylsulfano, or other functionality. A “primary amino”group is —NH₂. “Mono-substituted amino” means a radical —N(H)Rsubstituted as above and includes, e.g., methylamino,(1-methylethyl)amino, phenylamino, and the like. “Di-substituted amino”means a radical —N(R)R′ substituted as above and includes, e.g.,dimethylamino, methylethylamino, di(1-methylethyl)amino, and the like.

Carboxyalkyl: A functional group with the formula —COOR where R isalkyl.

DiA: 4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide

HPLC: high performance (or pressure) liquid chromatography

LPA: lysophosphatidic acid

LPS: lysophosphatidyl serine

PA: phosphatidic acid

PC: phosphatidyl choline

PE: phosphatidylethanolamine

PI: phosphatidyl inositol

Probe: A substance used to detect or identify another substance in asample.

S1P: sphingosine-1-phosphate

SPE: solid phase extraction

Substituent: An atom or group of atoms that replaces another atom in amolecule as the result of a reaction. The term “substituent” typicallyrefers to an atom or group of atoms that replaces a hydrogen atom on aparent hydrocarbon chain or ring.

Substituted: A fundamental compound, such as an aryl or aliphaticcompound, or a radical thereof, having coupled thereto, typically inplace of a hydrogen atom, a second substituent. For example, substitutedaryl compounds or substituents may have an aliphatic group coupled tothe closed ring of the aryl base, such as with toluene. Again solely byway of example and without limitation, a long-chain hydrocarbon may havea substituent bonded thereto, such as one or more halogens, an arylgroup, a cyclic group, a heteroaryl group or a heterocyclic group.

II. OVERVIEW OF VARIOUS EMBODIMENTS

Embodiments of compounds for detecting lysophosphatidic acids have astructure according to any one of general formulas I-IV. When thecompound has general formula I, R¹-R⁶ independently are hydrogen,hydroxyl, thiol, lower alkyl, carboxyalkyl, amino, lower alkoxy, orhalogen; R⁷-R¹⁰ independently are hydrogen, alkyl, acyl, carboxyl,nitro, amino, alkylamino, or —SO₃H; R¹¹ is N—C(═R¹³)—NH₂,N—NH—C(═R¹³)—NH₂, N—C(NH₂)═N—C(═R¹³)—NH₂, or N—NH—C(NH₂)═N—C(═R¹³)—NH₂,where R¹³ is O, S, or NH; each R¹² independently is hydrogen or loweralkyl, or each of R¹, R², R⁵, and R⁶ may together with an adjacent R¹²and N atom form a 6-membered heterocyclic ring; and X is O, S, CH₂, NH,or SiR¹⁴ where R¹⁴ is H or lower alkyl. In some embodiments R¹-R¹⁰ areH.

When the compound has general formula II, R¹-R⁵, R¹⁵, R¹⁷, and R¹⁸independently are hydrogen, hydroxyl, thiol, lower alkyl, carboxyalkyl,amino, lower alkoxy, or halogen; R¹⁶ is —N(R¹²)₂; R⁷-R¹⁰ independentlyare hydrogen, alkyl, acyl, carboxyl, nitro, amino, alkylamino, or —SO₃H;R¹¹ is N—C(═R¹³)—NH₂, N—NH—C(═R¹³)—NH₂, N—C(NH₂)═N—C(═R¹³)—NH₂, orN—NH—C(NH₂)═N—C(═R¹³)—NH₂, where R¹³ is O, S, or NH; each R¹²independently is hydrogen or lower alkyl, or each of R¹, R², R¹⁵, andR¹⁷ may together with an adjacent R¹² and N atom form a 6-memberedheterocyclic ring; and X is O, S, CH₂, NH, or SiR¹⁴ where R¹⁴ is H orlower alkyl. In some embodiments, R¹-R⁵, R⁷-R¹⁰, R¹⁵, R¹⁷, and R¹⁸ arehydrogen.

When the compound has general formula III, R²-R⁵, R¹⁵, R¹⁷, and R¹⁹-R²¹independently are hydrogen, hydroxyl, thiol, lower alkyl, carboxyalkyl,amino, lower alkoxy, or halogen; one of R¹⁶ and R¹⁸ is hydrogen,hydroxyl, thiol, lower alkyl, carboxyalkyl, amino, lower alkoxy, orhalogen, and the other of R¹⁶ and R¹⁸ is —N(R¹²)₂; R⁷-R¹⁰ independentlyare hydrogen, alkyl, acyl, carboxyl, nitro, amino, alkylamino, or —SO₃H;R¹¹ is N—C(═R¹³)—NH₂, N—NH—C(═R¹³)—NH₂, N—C(NH₂)═N—C(═R¹³)—NH₂, orN—NH—C(NH₂)═N—C(═R¹³)—NH₂, where R¹³ is O, S, or NH; each R¹²independently is hydrogen or lower alkyl, or if R¹⁶ is —N(R¹²)₂, each ofR¹⁵, R¹⁷, R²⁰, and R²¹ may together with an adjacent R¹² and N form a6-membered heterocyclic ring; and X is O, S, CH₂, NH, or SiR¹⁴ where R¹⁴is H or lower alkyl. In some embodiments, R²-R⁵, R⁷-R¹⁰, R¹⁵, R¹⁷, andR¹⁹-R²¹ are hydrogen, one of R¹⁶ and R¹⁸ is hydrogen, and the other ofR¹⁶ and R¹⁸ is —N(R¹²)₂.

When the compound has general formula IV, R¹-R⁴, R⁶, and R²²-R²⁴independently are hydrogen, hydroxyl, thiol, lower alkyl, carboxyalkyl,amino, lower alkoxy, or halogen; R⁷-R¹⁰ independently are hydrogen,alkyl, acyl, carboxyl, nitro, amino, alkylamino, or —SO₃H; R¹¹ isN—C(═R¹³)—NH₂, N—NH—C(═R¹³)—NH₂, N—C(NH₂)═N—C(═R¹³)—NH₂, orN—NH—C(NH₂)═N—C(═R¹³)—NH₂, where R¹³ is O, S, or NH; each R¹²independently is hydrogen or lower alkyl, or each of R¹, R², R²³, andR²⁴ may together with an adjacent R¹² and N form a 6-memberedheterocyclic ring; and X is O, S, CH₂, NH, or SiR¹⁴ where R¹⁴ is H orlower alkyl. In some embodiments, R¹-R⁴, R⁶⁻¹⁰, and R²²-R²⁴ arehydrogen.

In any or all of the above embodiments, each R¹² independently may bemethyl or ethyl. In any or all of the above embodiments, X may beoxygen.

In some embodiments, the compound is

A kit for detecting and quantifying lysophosphatidic acid comprises atleast one compound according to any or all of the above embodiments. Insome embodiments, the kit further comprises one or more lysophosphatidicacid species. In any or all of the above embodiments, the kit mayfurther comprise one or more solid-phase extraction cartridges whereinthe stationary phase is C8. In any or all of the above embodiments, thecompound may be

A method for quantifying lysophosphatidic acid species comprisescombining a sample that may include one or more lysophosphatidic acidspecies with a compound according to any or all of the above embodimentsin a solvent comprising dimethylsulfoxide in methanol to form asolution; exposing the solution to a light source; measuringfluorescence intensity of the solution; and determining, based on thefluorescence intensity, a total concentration of lysophosphatidic acidspecies in the sample. The solvent may comprise 2.5-10%dimethylsulfoxide in methanol. In some embodiments, the sample isobtained by extracting lysophosphatidic acid species from a plasma orserum sample.

A method for extracting lysophosphatidic acid species includes combininga sample of plasma or serum with a solvent comprising a lower alkylalcohol and a relatively nonpolar solvent, e.g., chloroform, to form amixture; separating organic and aqueous layers of the mixture;extracting the aqueous layer with a buffer at neutral pH to form anextracted aqueous phase; mixing the extracted aqueous phase withchloroform; separating chloroform from the extracted aqueous phase toform a washed aqueous phase; adding phosphoric acid to the washedaqueous phase to form an acidified aqueous phase; loading the acidifiedaqueous phase onto a solid-phase extraction (SPE) cartridge including astationary phase comprising silica derivatized with hydrocarbon chains;flowing water and subsequently chloroform through the SPE cartridge;drying the SPE cartridge; and flowing a lower alkyl alcohol through theSPE cartridge, thereby eluting lysophosphatidic acid species in thelower alkyl alcohol from the SPE cartridge.

In some embodiments, combining the plasma or serum sample with thesolvent comprises combining one part of the plasma or serum sample withfive parts of the solvent, the solvent comprising methanol andchloroform in a ratio of 2:1. In any or all of the above embodiments,the method may further comprise incubating the mixture at 4° C. for aperiod of time; and warming the mixture to ambient temperature beforeseparating the organic and aqueous layers of the mixture. In any or allof the above embodiments, extracting the aqueous layer with a buffer atneutral pH may comprise extracting with phosphate-buffered saline at pH7.4. In any or all of the above embodiments, the steps of mixing theextracted aqueous phase with chloroform and separating chloroform fromthe extracted aqueous phase to form a washed aqueous phase may berepeated. In any or all of the above embodiments, sufficient phosphoricacid may be added to the washed aqueous phase to reduce pH to 2. In anyor all of the above embodiments, the stationary phase of the SPEcartridge may comprise silica derivatized with octyl chains. In any orall of the above embodiments, the lower alkyl alcohol may be methanol.In any or all of the above embodiments, the method may further includeevaporating the lower alkyl alcohol to form a dry residue comprisinglysophosphatidic acid species; and dissolving the dry residue in 9:1methanol:H₂O to produce an extracted lysophosphatidic acid samplecomprising one or more lysophosphatidic acid species. In someembodiments, the method further includes determining a totalconcentration of lysophatidic acid species in the extractedlysophosphatidic acid sample.

In some embodiments, the method further includes determining a totalconcentration of lysophatidic acid species by combining the extractedlysophosphatidic acid sample with a compound as disclosed herein in asolvent comprising dimethylsulfoxide in methanol to form a solution;exposing the solution to a light source; measuring fluorescenceintensity of the solution; and determining, based on the fluorescenceintensity, the total concentration of lysophosphatidic acid species. Thesolvent may comprise 2.5-10% dimethylsulfoxide in methanol.

In some embodiments, the compound used for determining a totalconcentration of lysophosphatidic acid species is

and fluorescence intensity is measured at 570 nm.

In any or all of the above embodiments, the sample may be obtained froma subject suspected of being at risk of a condition associated with anaberrant LPA level, the method further comprising determining a risklevel for the condition, wherein the risk level is based at least inpart on the total concentration. In some embodiments, the condition iscancer, e.g., ovarian cancer, cardiovascular disease, plateletaggregation, ischemia perfusion injury, neuropathic pain, aneuropsychiatric disorder, a reproductive disorder, or fibrosis.

In some embodiments, the method further includes separatinglysophosphatidic acid species in the extracted lysophosphatidic acidsample using a reversed-phase high-performance liquid chromatography(HPLC) column, and detecting individual lysophosphatidic acid species asthe separated lysophosphatidic acid species exit the reversed-phase HPLCcolumn. Separating lysophosphatidic acid species using a reversed-phasehigh-performance liquid chromatography (HPLC) column may furthercomprise flowing the sample into the reversed-phase HPLC column, whereinthe column has a C8 stationary phase, and subsequently flowing 16:5methanol/phosphate buffer (50 mM, pH 2.5) through the reversed-phaseHPLC column, thereby forming an eluent comprising lysophosphatidic acidspecies. In any or all of the above embodiments, detecting individuallysophosphatidic acid species may comprise combining the eluent with4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide (DiA) as itexits the reversed-phase HPLC column to form a DiA-eluent mixture;flowing the DiA-eluent mixture through a detector; and detectingindividual lysophosphatidic acid species by detecting fluorescence ofthe DiA-eluent mixture as the DiA-eluent mixture flows through thedetector. In some embodiments, the method further comprises identifyingan individual lysophosphatidic acid species by comparing an elution timefor the individual lysophosphatidic acid species to elute from thereversed-phase HPLC column to elution times for known individuallysophosphatidic acid species, measuring fluorescence intensity of theDiA-eluent mixture, and determining, based on the fluorescenceintensity, a concentration of the individual lysophosphatidic acidspecies.

In any or all embodiments of the above methods, the plasma or serumsample may be obtained from a subject suspected of being at risk of acondition associated with an aberrant LPA level, the method furthercomprising determining a risk level for the condition, wherein the risklevel is based at least in part on an identification of an individuallysophosphatidic acid species, the concentration of the individuallysophosphatidic acid species, or a combination thereof. In someembodiments, the condition is cancer, e.g., ovarian cancer,cardiovascular disease, platelet aggregation, ischemia perfusion injury,neuropathic pain, a neuropsychiatric disorder, a reproductive disorder,or fibrosis.

III. LPA ISOLATION

LPAs are found in plasma and serum. To accurately quantify total LPAand/or to separate, identify, and quantify LPA species, the LPAs must beextracted from the plasma or serum sample and separated from potentialinterferences, including other phospholipids (e.g., phosphatidic acids,phosphatidyl cholines, lysophophatidyl cholines, lysophosphatidylserines, phosphatidyl ethanolamine, phosphatidyl inositol, andsphingosine-1-phosphate) that may interfere.

Conventional methods for extracting LPAs insufficiently removeinterferences and/or produce poor LPA recoveries. These methods do notadequately prepare LPA samples for quantifying total LPA, or for HPLCseparation and subsequent identification/quantification of individualLPA species as disclosed herein. For example, solid phase extraction(SPE) is an accepted method for the removal of potential interferencesfrom biological samples, and has been used for the isolation andenrichment of the different classes of phospholipids. However, SPE alonedoes not sufficiently remove other phospholipids that can interfere withLPA separation and detection by HPLC. Additionally, although hybridzirconia and titania support cartridges are known to be useful forrecovery of phosphopeptides and removal of phospholipids from biologicalmedia, poor recoveries of LPAs were obtained under all evaluatedconditions. Because SPE does not sufficiently remove interferingphospholipids, some reported methods include a liquid-liquid extractionstep. Typical procedures require plasma acidification prior toextraction. However, evaluation of these methods demonstrated very lowLPA recoveries, and insufficient removal of other interferences.

Embodiments of the disclosed method for LPA extraction include aliquid-liquid extraction followed by reversed-phase SPE. Liquid-liquidextraction may be performed with a solvent mixture comprising a loweralkyl alcohol (e.g., methanol, ethanol) and a relatively nonpolarsolvent. In some examples, a solvent comprising methanol and chloroform,such as 2:1 MeOH:CHCl₃, is mixed with a plasma or serum sample in aratio of five parts solvent to one part plasma/serum. The combinedsolvent mixture and plasma/serum sample is incubated at 4° C. for aneffective period of time, and then warmed to ambient temperature (e.g.,20° C.). In some embodiments, the effective period of time is at least15 minutes, such as 15-30 minutes. In certain examples, the effectiveperiod of time is 20 minutes. The organic and aqueous layers areseparated (e.g., by centrifugation), and the upper aqueous layer isrecovered.

Phosphatidic acids are negatively charged at neutral pH. Thus,extraction at neutral pH selectively removes negatively charged LPAsfrom other neutral and positively charged phospholipids. Accordingly,the upper aqueous layer is extracted with a buffer near physiologic pH,such as phosphate-buffered saline (10 mM, pH 7.4). The aqueous phase iswashed with chloroform, and then acidified with phosphoric acid to pH 2.

Reversed-phase SPE separates compounds based on polarity. An SPE columnincludes a stationary phase (e.g., silica) derivatized with hydrocarbonchains. A solution comprising a mixture of compounds with varyingpolarities is passed through the column. Compounds with low-polarity areretained on the stationary phase while more polar compounds elute withthe solvent. The less polar compounds then are eluted from thestationary phase using a nonpolar solvent. In some embodiments, a C8 SPEcartridge is used. A C8 stationary phase comprises silica functionalizedwith octyl chains. The cartridge may be preconditioned with a polarsolvent, or series of polar solvents, such as methanol followed bywater. The acidified aqueous phase from the liquid:liquid extraction isloaded onto the cartridge. The cartridge is washed first with water toremove any polar species, and then with chloroform. The SPE cartridge isdried, with a nitrogen stream, to remove residual chloroform, and LPAssubsequently are eluted with a lower alkyl alcohol, e.g., methanol. Themost hydrophobic species are retained on the stationary phase.

In certain examples, isolating LPA includes obtaining a plasma or serumsample, e.g., from a subject having, or being at risk of developing, acondition associated with aberrant LPA levels. The plasma or serumsample is combined with a solvent comprising 2:1 methanol:chloroform;the combined mixture includes one part plasma or serum sample and fiveparts solvent. In certain examples, 0.8 mL of plasma or serum sample and4 mL of solvent are used. The mixture is thoroughly mixed, e.g., byvortexing at 2,000 rpm for 30 seconds. The mixture is then incubated at4° C. for 20 minutes, warmed to ambient temperature, and the aqueous andorganic layers are separated, e.g., by centrifugation at 2000 rpm for 10minutes. The upper aqueous layer is extracted with 2 mLphosphate-buffered saline (10 mM, pH 7.4) by vortexing at 2000 rpm for30 seconds. The aqueous phase is separated and washed twice withchloroform (1.33 mL). After each washing, the layers are separated, andthe chloroform layer is discarded. The washed aqueous phase is acidifiedto pH 2 with phosphoric acid. The acidified aqueous phase is then loadedon a C8 SPE cartridge that has been preconditioned with 6 mL ofmethanol, followed by 3 mL of water. After the sample is loaded onto thecartridge, the cartridge is rinsed with 3 mL water, and then 1 mLchloroform. The cartridge then is dried, e.g., with a nitrogen stream.LPAs are eluted from the dried cartridge with 4 mL methanol. The elutedLPAs may be dried, and reconstituted in 9:1 methanol:water for furtheranalysis, such as HPLC separation.

IV. LPA SPECIES HPLC SEPARATION AND POST-COLUMN DETECTION

Following solid-phase extraction of LPAs from plasma or serum, the LPAspecies are separated using HPLC and then detected. A fluorescent probe4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide (DiA, below) isused as a post-column reagent for the fluorescent detection andquantification of phospholipids. DiA fluorescence can be detected withan excitation wavelength of 450-470 nm and an emission wavelength of570-590 nm. Embodiments of the disclosed method, including the initialLPA extraction, are capable of separating and quantifying at least sixindividual LPA species. In some embodiments, six individual LPA speciesat physiological levels in plasma can be separated and detected in 15minutes. Compared to conventional LC-MS methods, embodiments of thedisclosed method are more practical and reliable since the method is notprone to ionization-related issues and is relatively inexpensive forroutine analysis.

HPLC can be used to separate LPA species. Desirably, HPLC separationwill provide complete separation of LPA species with minimal peakbroadening and a reasonable separation time (e.g., less than 20minutes). It was determined that reversed-phase HPLC would be suitable.An extensive evaluation of solvent systems determined that commonsolvent mixtures (methanol/water, acetonitrile/water) did not adequatelyresolve LPA species. Phosphate buffer was found to provide the bestresults. Complete separation of LPA species depends, at least in part,on buffer concentration and pH, and optimal separation was obtained with50 mM phosphate buffer, pH 2.5. In some examples, LPAs were eluted usinga 16:5 mixture of methanol:phosphate buffer (50 mM, pH 2.5).

Several reversed-phase columns were evaluated, and C8-based columns werefound to provide superior results. A shorter column (i.e., 50 mm vs. 100mm) minimized peak broadening and provided a desirably short separationtime of ˜15 minutes. Peak width is a result-effective variable, withnarrower peaks providing better separation (less diffusion and overlapof adjacent peaks) and allowing a lower detection limit due to lessdilution of the LPA species.

In some embodiments, the LPA species are detected with DiA. DiA is mixedwith the eluent exiting the HPLC column (e.g., via a 3-way connector ormixing tee), and the mixture then flows into a fluorescence detector. Asindividual LPA species exit the column, become mixed with DiA, and flowinto the detector, fluorescence increases and the LPA species aredetected. DiA concentration and flow rate are both result-effectivevariables. Both concentration and flow rate are selected to provide anoptimal signal-to-noise ratio with a relatively low fluorescencebackground. In some examples, a 10 μM aqueous DiA solution with a flowrate of 0.60 mL/minute to 0.65 mL/minute (e.g., 0.62 mL/min.) was used.The lower limit of LPA detection was determined as being the amount ofan LPA species that produced a signal-to-noise ratio of 3:1. Embodimentsof the disclosed method have a lower limit of detection of ≤0.3 μM, suchas ≤0.2 μM or ≤0.1 μM. Solutions of known LPA species can be used todetermine retention times for identification of peaks.

LPA species can be quantified by first preparing calibration curvesusing standard solutions of LPA species at known concentrations. BecauseDiA fluorescence intensity varies for different LPA species atequivalent concentrations, a calibration curve is prepared for eachspecies. Once the calibration curves are prepared, the concentrations ofLPA species in an unknown sample can be determined by comparingfluorescence intensity of each LPA species to the respective calibrationcurve.

In certain embodiments, 20 μL of extracted LPAs in 9:1 methanol:water isinjected onto a C8-based HPLC column (e.g., LUNA™ C8 column, 50×2 mm, 3μm) and eluted with a solvent comprising 16:5 methanol:phosphate buffer(50 mM, pH 2.5) at a flow rate of 0.32 mL/minute. The distal end of theHPLC column is coupled to one inlet of a mixing tee. An aqueous DiAsolution (10 μM) is coupled to a second inlet of the mixing tee and setto a flow rate of 0.62 mL/minute. The combined eluent and DiA solutionflows through the outlet of the mixing tee and into a fluorescencedetector. Fluorescence is measured using an excitation wavelength of 450nm and an emission wavelength of 570 nm. As each LPA species elutes fromthe HPLC column and flows through the detector, an increase influorescence is observed.

Identification and quantification of individual LPA species can be usedto evaluate a subject's risk level of having, or developing, a conditioncorrelated with aberrant LPA levels. In some embodiments, a subject'srisk of having or developing an LPA-related condition is proportional tothe concentration of one or more LPA species in the subject's blood. Forexample, elevated levels of plasma LPA have been found in patients withovarian cancer. Other conditions associated with elevated LPA levelsinclude other cancers (e.g. breast cancer, gynecological cancers),cardiovascular disease, platelet aggregation, ischemia perfusion injury,wound healing, neuropathic pain, neuropsychiatric disorders,reproductive disorders, and fibrosis. Some conditions are correlatedwith elevated levels of only certain LPA species. Thus, determining thepresence and concentration of individual LPA species may improvediagnosis accuracy.

V. COMPOUNDS AND METHODS FOR TOTAL LPA DETECTION

In some conditions, total LPA concentration may be predictive of asubject's risk of having, or developing, a condition associated withaberrant LPA levels. Thus, it is desirable to have a single probecapable of detecting a plurality of LPA species with equivalentsensitivity.

Fluorogenic probes commercially available for phospholipid detection canbe classified as fluorescent lipid analogs. Phospholipid detection isbased on the enhancement or quenching of fluorescence emission intensityas a result of aggregation/de-aggregation between the phospholipid andthe fluorophore. Since this non-specific process is dependent on thetype, length and number of alkyl chains present in the phospholipid;these probes are not useful for total phospholipid analysis since thefluorescence intensity will vary from phospholipid to phospholipid.Thus, if a plurality of phospholipids is present, the concentrationcannot be accurately determined based upon the total fluorescence.

Embodiments of the disclosed fluorogenic compounds are based on arhodamine B framework, and are functionalized with a guanidine,biguanidine, guanylurea, or guanylthiourea group. Upon interaction ofthe guanidine, biguanidine, guanylurea, or guanylthiourea group with thephosphate group of a phospholipid, electrostatic interactions will alterthe compound's fluorescence.

Some embodiments of the disclosed compounds have a structure accordingto general formula I.

In general formula I, R¹-R⁶ independently are hydrogen, hydroxyl, thiol,lower alkyl, carboxyalkyl, amino, lower alkoxy, or halogen; R⁷-R¹⁰independently are hydrogen, alkyl, acyl, carboxyl, nitro, amino,alkylamino, or —SO₃H; R¹¹ is N—C(═R¹³)—NH₂, N—NH—C(═R¹³)—NH₂,N—C(NH₂)═N—C(═R¹³)—NH₂, or N—NH—C(NH₂)═N—C(═R¹³)—NH₂, where R¹³ is O, S,or NH; each R¹² independently is hydrogen or lower alkyl, or each of R¹,R², R⁵, and R⁶ may together with an adjacent R¹² and N atom form a6-membered heterocyclic ring; and X is O, S, CH₂, NH, or SiR¹⁴ where R¹⁴is H or lower alkyl. In some embodiments, each R¹² independently ismethyl or ethyl. In certain embodiments, R¹-R¹⁰ are hydrogen.

In some embodiments, the disclosed compounds have a structure accordingto general formula II.

In general formula II, R¹-R⁵, R¹⁵, R¹⁷, and R¹⁸ independently arehydrogen, hydroxyl, thiol, lower alkyl, carboxyalkyl, amino, loweralkoxy, or halogen; R¹⁶ is N(R¹²)₂; R⁷-R¹⁰ independently are hydrogen,alkyl, acyl, carboxyl, nitro, amino, alkylamino, or —SO₃H; R¹¹ isN—C(═R¹³)—NH₂, N—NH—C(═R¹³)—NH₂, N—C(NH₂)═N—C(═R¹³)—NH₂, orN—NH—C(NH₂)═N—C(═R¹³)—NH₂, where R¹³ is O, S, or NH; each R¹²independently is hydrogen or lower alkyl, or each of R¹, R², R¹⁵, andR¹⁷ may together with an adjacent R¹² and N atom form a 6-memberedheterocyclic ring; and X is O, S, CH₂, NH, or SiR¹⁴ where R¹⁴ is H orlower alkyl. In some embodiments, each R¹² independently is methyl orethyl. In certain embodiments, R¹-R⁵, R⁷-R¹⁰, R¹⁵, R¹⁷, and R¹⁸ arehydrogen.

Some embodiments of the disclosed compounds have a structure accordingto general formula III.

In general formula III, R²-R⁵, R¹⁵, R¹⁷, and R¹⁹-R²¹ independently arehydrogen, hydroxyl, thiol, lower alkyl, carboxyalkyl, amino, loweralkoxy, or halogen; one of R¹⁶ and R¹⁸ is hydrogen, hydroxyl, thiol,lower alkyl, carboxyalkyl, amino, lower alkoxy, or halogen, and theother of R¹⁶ and R¹⁸ is —N(R¹²)₂; R⁷-R¹⁰ independently are hydrogen,alkyl, acyl, carboxyl, nitro, amino, alkylamino, or —SO₃H; R¹¹ isN—C(═R¹³)—NH₂, N—NH—C(═R¹³)—NH₂, N—C(NH₂)═N—C(═R¹³)—NH₂, orN—NH—C(NH₂)═N—C(═R¹³)—NH₂, where R¹³ is O, S, or NH; each R¹²independently is hydrogen or lower alkyl, or if R¹⁶ is —N(R¹²)₂, each ofR¹⁵, R¹⁷, R²⁰, and R²¹ may together with an adjacent R¹² and N form a6-membered heterocyclic ring; and X is O, S, CH₂, NH, or SiR¹⁴ where R¹⁴is H or lower alkyl. In some embodiments, each R¹² independently ismethyl or ethyl. In certain embodiments, R²-R⁵, R⁷-R¹⁰, R¹⁵, R¹⁷,R¹⁹-R²¹, and one of R¹⁶ and R¹⁸ are hydrogen.

Some embodiments of the disclosed compounds have a structure accordingto general formula IV.

In general formula IV, R¹-R⁴, R⁶, and R²²-R²⁴ independently arehydrogen, hydroxyl, thiol, lower alkyl, carboxyalkyl, amino, loweralkoxy, or halogen; R⁷-R¹⁰ independently are hydrogen, alkyl, acyl,carboxyl, nitro, amino, alkylamino, or —SO₃H; R¹¹ is N—C(═R¹³)—NH₂,N—NH—C(═R¹³)—NH₂, N—C(NH₂)═N—C(═R¹³)—NH₂, or N—NH—C(NH₂)═N—C(═R¹³)—NH₂,where R¹³ is O, S, or NH; each R¹² independently is hydrogen or loweralkyl, or each of R¹, R², R²³, and R²⁴ may together with an adjacent R¹²and N form a 6-membered heterocyclic ring; and X is O, S, CH₂, NH, orSiR¹⁴ where R¹⁴ is H or lower alkyl. In some embodiments, each R¹²independently is methyl or ethyl. In certain embodiments, R¹-R⁴, R⁶,R⁷-R¹⁰, and R²²-R²⁴ are hydrogen.

Exemplary compounds include, but are not limited to:

Compounds according to general formulas I-IV include a lactam ring. Whenthe lactam ring is present, the compound is non-fluorescent. However,when the guanidine group interacts with the phosphate group oflysophosphatidic acid, the bond between R¹¹ and the upper 3-ring portionof the structure is broken and the lactam ring opens, thereby alteringelectrostatic interactions within the compound, and rendering thecompound fluorescent.

Some embodiments of compounds according to general formulas I-IV aresuitable for universal detection of LPA. In other words, substantiallythe same fluorescence intensity is obtained at a given LPA concentrationirrespective of the particular LPA species present. In some embodiments,when the compound is combined with a sample comprising isolated LPAs,the fluorescence intensity is stable over time. For example, thefluorescence intensity may remain substantially constant for at least 10minutes, at least 20 minutes, or at least 30 minutes.

A sample that may include one or more LPAs is combined with a compoundaccording to any one of general formulas I-IV in a suitable solvent toform a solution. The solution is exposed to a light source, and LPAs, ifpresent, are detected by detecting fluorescence of the solution at asuitable wavelength. For GRBI and GRBII, an excitation wavelength of 550nm and an emission wavelength of 570 nm may be used. Suitable solventsinclude those in which both the compound and the LPAs are soluble.Exemplary solvents include chloroform/DMSO mixtures, such as mixturescomprising 2.5-10% (v/v) DMSO. In certain examples, a sample potentiallycomprising LPAs is combined with a compound according to any one ofgeneral formulas I-IV in a solvent comprising 9:1 chloroform:DMSO.Desirably, the compound is present in an excess amount compared to theLPAs.

In some embodiments, the LPA sample is obtained from a biologicalsample, e.g., plasma or serum obtained from a subject at risk of having,or developing, a condition correlated with aberrant LPA levels. The LPAsample may be prepared from the biological sample using liquid-liquidextraction followed by SPE extraction as described herein.

A calibration curve can be prepared by combining a compound according toany one of general formulas I-IV with a series of samples having knownconcentrations of one or more LPA species and measuring the fluorescenceintensity of each solution. The concentration of LPA in an unknownsample is determined by combining the unknown sample with the compound,measuring the fluorescence intensity, and comparing the fluorescenceintensity to the calibration curve.

VI. SAMPLE COLLECTION AND STORAGE

Embodiments of the methods and compounds disclosed herein are suitablefor detecting LPAs in plasma and serum samples. Blood samples are storedat 4° C. before processing, such as for 4-8 hours. An anti-coagulant,e.g., EDTA, is added to plasma samples before storage to preventplatelet aggregation and inhibit cation-dependent lipid enzymaticprocesses. Platelets are removed from plasma samples before storage;otherwise, the platelets may undergo lysis and release additionalamounts of LPA into the sample. The stored samples are centrifuged toremove blood cells before analysis. In some embodiments, the sample iscentrifuged at 1750×g for 15 minutes at ambient temperature (˜20° C.).The supernatant (plasma or serum) is stored in low-binding siliconizedtubes or glass tubes. (Baker et al., JAMA, J. Am. Med. Assoc., 2002,287:3081-3082; Xiao et al., Ann. N. Y. Acad. Sci., 2000, 905:242-259; Xuet al., JAMA, J. Am. Med. Assoc., 1998, 280:719-723; Yi et al.,Functional Lipidomics, CRC Press, 2005, 125-146). Desirably, sufficientblood is collected to provide a plasma or serum sample with a volume ofat least 3.5 mL, or at least 5 mL, such that the sample may be evaluatedin triplicate.

VII. KITS

Kits for total LPA quantification are also a feature of this disclosure.Embodiments of the kits include at least one compound according to anyone of general formulas I-IV and suitable for detecting LPAs extractedfrom a sample (e.g., plasma or serum). In some embodiments, the probe isGRBI or GRBII. In some embodiments, the kits also include one or moresolvents suitable for detecting fluorescence of LPAs when combined withthe probe. For example, the kit may include chloroform anddimethylsulfoxide, or a solution comprising 9:1 chloroform:DMSO.

The kits also may include one or more containers, such as a disposabletest tube or cuvette, in which the detection can be performed. Incertain embodiments, the compound may be premeasured into the one ormore containers, and the detection is subsequently performed by addingthe solvent(s) and test sample to the container).

The kits may further include instructions for performing the detectionand, optionally, instructions for extracting LPAs from a biologicalsample. The kits also may include one or more solid-phase extractioncartridges suitable for the extraction, e.g., one or more C8 SPEcartridges. In certain embodiments, the kit further includes one or morecontrol samples of LPAs. The control samples may be provided in solidform or in solution.

VIII. EXAMPLES Materials

All the lysophosphatidic acids including1-myristoyl-2-hydroxy-sn-glycero-3-phosphate (LPA 14:0),1-palmitoyl-2-hydroxy-sn-glycero-3-phosphate (LPA 16:0),1-heptadecanoyl-2-hydroxy-sn-glycero-3-phosphate (LPA 17:0),1-stearoyl-2-hydroxy-sn-glycero-3-phosphate (LPA 18:0),1-oleoyl-2-hydroxy-sn-glycero-3-phosphate (LPA 18:1) and1-arachidonoyl-2-hydroxy-sn-glycero-3-phosphate (LPA 20:4) werepurchased from Avanti Polar Lipids (Alabaster, Ala., USA).4-(4-(Dihexadecylamino)-styryl)-N-methylpyridinium iodide (DiA) waspurchased from AnaSpec (Fremont, Calif., USA). HPLC grade MeOH waspurchased from Fisher Scientific. Ultra-pure water was obtained from aMILLI-Q™ ultra-pure water system. Phosphoric acid and monosodiumphosphate were purchased from Sigma-Aldrich (St. Louis, Mo., USA).Waters OASIS™ HLB (3 cc, 60 mg, 30 μm) SPE cartridges were purchasedfrom Waters Corporation (Milford, Mass., USA).

Rhodamine B, Rhodamine B Base, 1,3-bis-Boc-2-methyl-2-thiopseudourea,Sodium methoxide solution (0.5 M), and1,3-bis(tert-butoxycarbonyl)guanidine were purchased from Aldrich.Anhydrous potassium carbonate and trifluoroacetic acid were purchasedfrom Fisher Scientific. Mercury(II) chloride was purchased from AcrosOrganics. LPA sodium salts were purchased from Avanti. Silica gel waspurchased from Sorbent Technologies. All chemicals were used as receivedwithout further purification.

Instrumentation:

Fluorescence measurements were performed on a Cary Eclipse fluorescencespectrophotometer and absorption spectra on a Cary 50 UV-Visspectrophotometer (Agilent Technologies).

The HPLC system consists of a 1525 binary HPLC delivery system, a 2475multi lambda fluorescence detector (Waters). A LUNA™ C8 (50×2 mm, 3 μm)column connected to a guard cartridge with 2.0 to 3.0 mm internaldiameters was used for all the separations (Phenomenex). The reagent ispumped by a reagent manager (Waters). The post-column reagent and HPLCsystem are mixed and delivered to the detector by a metal mixing tee.The data was collected and processed with the EMPOWER™ software suite(Waters).

For LC/ESI/MS/MS, LPAs were separated in an ACCELA® UPLC system (ThermoFisher, San Jose, Calif.) and detected in an LTQ-ORBITRAP™ XL DISCOVERY®instrument (San Jose, Calif., USA), equipped with an ESI ion masssource. The data was collected in negative mode and processed with theXCALIBUR® software suite.

NMR spectra were recorded on a Bruker spectrometer in CDCl₃ and DMSO-d₆solutions, and chemical shifts were reported in δ units.

High-resolution ESI/MS spectra were recorded on a ThermoElectronLTQ-ORBITRAP™ DISCOVERY® instrument equipped with an ESI ion masssource.

Example 1 Extraction of LPAs

Several solid supports were initially evaluated in LPA control mixturescontaining LPA 14:0, 16:0, 17:0, 18:0, 18:1 and 20:4. Despite the commonuse of hybrid Zr and TiO₂ support cartridges for the recovery andenrichment of phosphopeptides and removal of phospholipids frombiological media, little or no LPA recovery was obtained under all thedifferent conditions tested.

Reversed-phase SPE materials provided better results. Three differentcommercial reversed-phase C8 SPE cartridges, including Waters (SEP-PAK®Plus C-8 cartridge, 200 mg, 37-55 μm), Supelco (DISCOVERY® DSC-8cartridge, 3 mL, 500 mg, 50 μm) and Waters (OASIS™ HLB cartridge 3 mL,60 mg, 30 μm) were evaluated. From the evaluated commercial cartridges,the OASIS™ HLB cartridge proved to be the best in terms of LPArecoveries.

A liquid-liquid extraction prior to the SPE procedure was desirable toremove other phospholipids that may interfere with the detection ofLPAs. Typical procedures reported in the literature involveacidification of plasma prior to a liquid-liquid extraction. However,this procedure gave very low LPA recoveries and removal of otherinterferences.

Control of pH was discovered to have an important role in selectiveremoval of interferences. Because phosphatidic acids have a pKa₁=2.9 andpKa₂=7.5 (Kooijman et al., Biochemistry, 2005, 44:17007), they arenegatively charged at neutral pH. Thus performing liquid-liquidextraction at this condition separated neutral and positively chargedphospholipids from the negatively-charged LPAs.

Acidification of the aqueous phase at pH 5.0, 4.0, 3.0 and 2.5 in theSPE step was evaluated, revealing that lower pH during SPE improved LPAenrichment. The use of MeOH as the final solvent allowed the selectiveelution of LPAs, leaving behind the most hydrophobic species. The bestrecoveries (74-100%) were obtained at pH 2.5.

Recovery tests were performed. Three concentrations including 0.25 μM, 3μM and 5 μM of each LPA species mixed in PBS buffer were tested afterliquid-liquid extraction and SPE. Each buffered LPA solution was mixedwith 4 mL of MeOH:CHCl₃ 2:1 and vortexed at 2000 rpm for 30 seconds. Themixture was incubated at 4° C. for 20 minutes and warmed to roomtemperature. The mixture was then centrifuged at 2000 rpm for 10minutes, and the upper layer was separated and extracted with 2 mL PBS(10 mM, pH 7.4) by vortexing at 2000 rpm for 30 seconds. The aqueousphase was separated and washed with 1.33 mL CHCl₃ twice, then acidifiedto pH 2 with concentrated H₃PO₄. An SPE cartridge was preconditionedwith 6 mL of MeOH, followed by 3 mL of H₂O. The sample was loaded to thecartridge and rinsed with 3 mL of H₂O and then with 1 mL of CHCl₃. TheSPE cartridge was dried by applying a N₂ stream, and LPAs were elutedwith 4 mL of MeOH. The MeOH was evaporated, and the residue wasreconstituted in 0.16 mL of MeOH:H₂O 9:1. Samples (20 μL) obtained fromthe SPE purification step were injected and eluted with a mixture ofMeOH:phosphate buffer (50 mM, pH 2.5) 16:5 through a C8 column. The endof the column was connected to a mixing tee allowing contact with thepost-column reagent solution (DiA, 10 μM). The flow rate of the mobilephase was set to 0.32 mL/min and 0.62 mL/min for the post-columnreagent. Each concentration was done three times.

As shown in Table 1, the recoveries of LPA 14:0, LPA 16:0, LPA 17:0, LPA18:0, LPA 18:1 and LPA 20:4 are 93.45%, 94.15%, 85.14%, 76.87%, 76.86%and 73.79%, respectively. In addition, the contribution of otherphospholipids that have been identified to be present in human plasmawas evaluated. These other phospholipids can result in potential falsepositives for LPAs. In general, it is known that phospholipids are proneto either chemical or enzymatic hydrolysis. Phosphatidic acids (PAs)hydrolyze producing their corresponding LPAs, adding to the apparent LPAconcentrations. Due to the acidic conditions in which the LPA solidphase extraction was carried out, control experiments were performed tomeasure at what extent PAs were hydrolyzed. The results demonstratedthat none of PA 14:0, PA 16:0, PA 18:0 or PA 18:1 was hydrolyzed.Phosphatidyl cholines (PCs) represent the major components of biologicalmembranes and are also prone to hydrolysis producing the correspondinglysophosphatidyl cholines (LPCs). It was determined that PCs do nothydrolyze under our enrichment conditions. On the other hand, LPCs andlysophosphatidyl serines (LPSs) were found to be potentialinterferences. While LPC interference was removed by doing threeconsecutive liquid-liquid extractions instead of one, the presence oflysophosphatidyl serines (LPS) was determined not to be significant,since they are usually found at a much lower concentration than LPAs.Other phospholipids including phosphatidyl ethanolamine (PE),phosphatidyl inositol (PI) and SIP were also evaluated and found not tobe interferences.

TABLE 1 Measured by Measured by HPLC-post column HPLC-MS/MS LPA Recoveryin RSD Recovery in RSD Species control (%) (%) control (%) (%) 14:093.45 4.8 93.67 2.9 20:4 73.79 4.3 76.60 1.2 16:0 94.15 5.4 95.72 4.618:1 76.86 4.9 77.55 1.7 17:0 85.14 3.8 84.97 4.6 18:0 76.87 4.4 73.142.4

Example 2 HPLC and Post-Column Procedure for LPA Separation andDetection

LPA Detection:

Fluorescent probes used for post-column detection of phospholipids,typically rely on non-covalent interactions of the phospholipid with theprobe's supramolecular assemblies that are solvent dependent. Examplesof these probes include 2,5-bis-2-(5-tert-Butyl)benzoxazolylthiophene(BBOT) and 1,6-diphenyl-1,3,5-hexatriene (DPH). Evaluation of BBOT andDPH produced higher fluorescence emission enhancement for double-chainphosphatidic acids (PA) compared to single-chain analogues(lysophosphatidic acids, LPAs) investigated herein. Another class offluorescent probes with amphiphilic properties was also evaluated.Although 10-N-nonyl acridine orange (NAO) has been proposed for theanalysis of specific phospholipids like cardiolipin (CL), it did notexhibit a significant spectral response for LPA. Extending the alkylchain of NAO to an octadecyl chain did not improve LPA detection.Evaluation of the amphiphilic cyanine type probe4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide (DiA) gave themost promising results for LPA detection. As shown in FIG. 1, 3 μM DiAshows an increase in absorbance at 440-450 nm in the presence of 10 μMLPA 18:0. DiA (3 μM) exhibits weak fluorescence emission at 590 nm(excitation: 470 nm) in aqueous medium, and upon addition of 10 μM LPA18:0, an increase in fluorescence emission is observed (FIG. 2).

To determine the linear response of DiA in the presence of LPA, aninitial evaluation using LPA (18:0) as the model compound was carriedout using direct fluorescence spectrometry. Solutions of increasedconcentrations (0, 6.25, 12.5, 18.75, 25, 37.5, 50, 75, 100 and 150 μM)of LPA (18:0) were prepared in a mixture of MeOH:CHCl₃ 1:1. To avoidaggregation of the lipids, films for each concentration tested wereprepared by evaporation under an Ar stream, and the films werereconstituted in MeOH. Choline chloride (final concentration 6.4 mM) wasadded before mixing with DiA (final concentration 2.67 μM) aqueoussolution. Emission spectra for each solution were collected from 480 to800 nm exciting at 470 nm (FIG. 3). As shown in the inset of FIG. 3, theplot of maximum fluorescence emission versus concentration demonstrateda good linear relationship (R²=0.997) between the fluorescence intensityand LPA (18:0) concentrations ranging from 1 to 16 μM.

LPA Separation:

The common solvent mixtures (MeOH/H₂O, MeCN/H₂O) used for reversed-phasechromatography were not able to resolve individual LPA species. From thevarious buffer systems evaluated, it was found that phosphate buffergave better separation and peak shapes. LPA species separation wasdependent on buffer pH and concentration. Optimal resolution wasachieved with 50 mM phosphate buffer pH 2.5. The use of mobile phasemodifiers, including trifluoroacetic acid (TFA) and choline chloridewere also evaluated. Only choline chloride gave suitable separation,however, it increased significantly both the time of analysis and columnback pressure.

Several reversed phase columns were evaluated. The best results wereobtained from C8-based columns. The DISCOVERY® BIO™ wide pore C8 column(100×2.1 mm, 3 μm) was able to separate all the LPAs well, but causedvery high pressure and limited optimization of the composition/flow rateof mobile phase, achieving only long analysis times (>40 minutes). TheLUNA™ C8 column (50×2 mm, 3 μm) allowed an increased flow rate, therebyproducing sharper peaks and significantly reducing the analysis time(˜15 minutes). The length of the tubing was also important for achievinggood peak resolution and lower limits of detection. Shorter tubingminimized peak broadening (and potential overlapping) due to diffusionas the LPA species flowed through the column. The effect of temperaturewas also studied when working with the longer tubing, with noappreciable change in the range 20-60° C.

Detection of Separated LPA Species:

The 3-D mode feature in the Waters 2475 fluorescence detector was usedto determine suitable excitation and emission wavelengths for detection.The detector gain was set to 100. The acquisition mode was set toexcitation scanning mode (330-530 nm) while keeping the emissionwavelength constant (570 nm).

Solutions of DiA with concentrations ranging from 3 to 20 μM were testedand compared. Among them, 10 μM showed optimal signal-to-noise ratiowith a relatively low fluorescence background. Reagent flow rates from0.15 to 0.70 mL/min were tested and compared. Higher flow rates resultedin higher signal to noise ratios; however, they also induced a weakersignal due to excessive dilution of the sample. The optimal reagent flowrate was found to be 0.62 mL/min.

FIG. 4 shows a representative trace for the separation and subsequentdetection of LPA 14:0, 16:0, 17:0, 18:0, 18:1 and 20:4 with DiA. An LPAmixture (10 μM LPA 14:0, 16:0, 18:0, 18:1, 20:4 and 20 μM LPA 17:0 as aninternal standard) was injected in a 20 μL injection loop.Chromatography conditions: (i) column: LUNA™ C8 column, 3 μm, 50×2.1 mm;(ii) mobile phase: MeOH:phosphate buffer (50 mM, pH 2.5) 16:5; (iii)flow rate: 0.32 mL/min.; (iv) injection volume: 20 μL; (v) sampleconcentration: 10 μM in MeOH:H₂O 9:1; (vi) post-column reagent: 10 μMDiA in H₂O; (vii) reagent flow rate: 0.62 mL/min; (viii) detectionwavelength: ex/em 450/570 nm.

Mixtures of LPAs (in MeOH:H₂O 9:1) with concentrations ranging from0.5-40 μM were evaluated using the same procedure to determine theconcentration range over which a linear response was obtained. LPA(17:0), a non-natural LPA, was added to these mixtures at aconcentration of 20 μM to act as an internal standard for furtherquantification. LPA 14:0, LPA 18:0 and LPA 18:1 demonstrated a linearresponse throughout this range, while LPA 16:0 and LPA 20:4 had a linearresponse over the range of 0.5-25 μM (FIGS. 5A-5E). Correlation factors(R²) >0.99 were obtained for all of the LPAs (Table 2). The limit ofdetection (LOD) for each LPA species was determined as the amount ofanalyte that corresponds to three times the signal of the backgroundnoise.

TABLE 2 Retention Linear LPA time range LOD species (min) (μM) R² (μM)14:0  3.50 0-40 0.9962 0.147 20:4  5.56 0-25 0.9960 0.161 16:0  6.640-25 0.9963 0.173 18:1  8.35 0-40 0.9949 0.074 18:0 13.75 0-40 0.99430.272

To make a comparison with the HPLC post-column method, the individualLPA species also were evaluated with LC/ESI/MS/MS. A concentration rangeof 0-40 μM was selected. LPA (17:0) was also used as an internalstandard. FIGS. 6A-E show calibration curves for the individual LPAspecies evaluated with LC/ESI/MS/MS. Acceptable correlation factors (R²)were obtained for all the LPAs (Table 3). The limit of detection (LOD)for each LPA species was determined as the amount of analyte thatcorresponds to three times the signal of the background noise

TABLE 3 Retention Linear LPA time range LOD specie (min) (μM) R² (μM)14:0  6.30 0-40 0.9982 0.0067 20:4  7.55 0-40 0.9981 0.0099 16:0  8.290-40 0.9986 0.0123 18:1  9.10 0-40 0.9989 0.0066 18:0 11.22 0-40 0.99850.0156

Two blind samples which included some of the LPA subspecies at differentconcentrations were prepared as above and tested in triplicate usingboth the HPLC post column method and LC/ESI/MS/MS. The LPAconcentrations were in agreement between the two methods and withinacceptable error as compared to the theoretical values (Table 4).

TABLE 4 Blind A Blind B Theo- Theo- retical retical Value LC/EST/ ValueLC/EST/ (μM) HPLC MS/MS (μM) HPLC MS/MS Concen- ExperimentalExperimental Concen- Experimental Experimental tration (μM) % error (μM)% error tration (μM) % error (μM) % error LPA 3.80 3.72 −2.11 3.81 0.26— — — — — 14:0 LPA 1.20 1.16 −3.33 1.21 0.83 0.80 0.86 7.50 0.72 −10.0016:0 LPA 2.50 2.34 −6.40 2.62 4.80 4.20 4.26 1.43 3.83 −8.81 18:0 LPA0.70 0.66 −5.71 0.71 1.43 — — — — — 18:1 LPA 4.90 4.83 −1.43 5.19 5.922.20 2.32 5.4.5 2.33 5.91 20:4

Example 3 Quantification of LPAs in Commercial Plasma and Commercial LPASpiked Plasma

Native LPA concentrations were determined in human plasma from 5different donors. Plasma Source for Donor A: Lyophilized Human Plasma,Sigma-Aldrich (Catalog # P9523). For donors B, C, D, and E, plasma wascollected by Lampire Biological Laboratories Inc., from female donors,processed to obtain platelet-free plasma, and frozen at −80° C. (Catalog#7303809).

The human plasma from these five donors was spiked with 0.5 μM of eachindividual LPA. Then, the individual LPA concentrations of these spikedplasma samples were determined. All the samples were analyzed bytriplicate using both HPLC-post column and LC/ESI/MS/MS methods.

HPLC-post column conditions: Column—reversed phase C8, 3 μm, 50×2.0 mm.Mobile phase—methanol:phosphate buffer (pH 2.5)=16:5. Flow rate—0.32mL/min. Injection volume—20 μL. Sample concentration—10 μM in MeOH:H₂O9:1. Post-column reagent—10 μM DiA in H₂O. Reagent flow rate—0.62mL/min. Detection wavelengths: ex/em 450/570 nm.

LC/ESI/MS/MS conditions: Column LUNA™ C8 column (50×2 mm, 3 μm) at 40°C. Injection volume—10 μL. Mobile phase-MeOH:aqueous formic acid (pH2.5) 9:1. Flow rate of 0.4 mL/min. Parent and daughter ions weredetected in the negative ion mode, sprayer voltage; 3.0 kV, capillarytemperature at 300° C.

The LPA concentrations determined by these two methods correlate witheach other (Tables 5A-5C). Acceptable standard deviations (a) wereobtained for each individual LPA for all donors: (a) HPLC-post columnmethod, σ: 0.002-0.091; (b) LC/ESI/MS/MS method, σ: 0.002-0.063. Theexperimental concentration determined for spiked samples is in agreementwith the actual spiked concentration. (a) HPLC-post column method: 0.406to 0.595 μM. (b) LC/ESI/MS/MS method: 0.370 to 0.592 μM.

FIG. 7 shows typical traces obtained by the HPLC-post column method fora standard LPA mixture and LPAs isolated from human plasma samples.FIGS. 8A and 8B show typical LC/ESI/MS/MS traces of a 10 μM mixture ofstandard LPAs (FIG. 8A) and a plasma sample from donor A (FIG. 8B).

Tables 5A-5C Summary of Results for LPA Analysis in Human Plasma Usingthe HPLC Post-Column Fluorescence and LC/ESI/MS/MS Methods

TABLE 5A Non Spiked Donor A Avg (σ)* Donor B Avg (σ)* Donor C Avg (σ)*Donor D Avg (σ)* Donor E Avg (σ)* HPLC HPLC HPLC HPLC HPLC Post- LC/ESIPost- LC/ESI Post- LC/ESI Post- LC/ESI Post- LC/ESI Column MS/MS ColumnMS/MS Column MS/MS Column MS/MS Column MS/MS LPA 0.90 0.92 0.97 1.030.76 0.68 0.24 0.23 0.17 0.18 14:0 (0.01) (0.01) (0.03) (0.01) (0.01)(0.02) (0.00) (0.01) (0.00) (0.01) LPA 0.63 0.64 0.98 0.94 0.21 0.270.26 0.28 0.20 0.23 20:4 (0.02) (0.03) (0.01) (0.01) (0.02) (0.02)(0..01) (0.01) (0.02) (0.01) LPA 0.76 0.74 0.96 1.04 0.55 0.42 0.45 0.430.29 0.28 16:0 (0.03) (0.01) (0.02) (0.02) (0.01) (0.04) (0.03) (0.01)(0.00) (0.02) 1,PA 0.68 0.65 1.05 1.03 0.37 0.32 0.30 0.38 0.53 0.4718:1 (0.01) (0.02) (0.00) (0.02) (0.01) (0.01) (0.02) (0.01) (0.01)(0.02) LPA 0.56 0.60 0.99 0.93 0.29 0.23 0.33 0.31 0.33 0.30 18:0 (0.02)(0.01) (0.01) (0.01) (0.03) (0.01) (0.02) (0.00) (0.00) (0.01) Total3.53 3.56 4.96 4.97 2.18 1.91 1.57 1.63 1.52 1.45 LPA (0.03) (0.02)(0.04) (0.04) (0.02) (0.09) (0.03) (0.03) (0.02) (0.05)

TABLE 5B Spiked with 0.5 μM LPA Donor A Avg (σ)* Donor B Avg (σ)* DonorC Avg (σ)* Donor D Avg (σ)* Donor E Avg (σ) HPLC HPLC HPLC HPLC HPLCPost- LC/ESI Post- LC/ESI Post- LC/ESI Post- CC/ESI Post- LC/ESI CohmmMS/MS Column MS/MS Column MS/MS Column MS/MS Column MS/MS LPA 1.31 1.331.43 1..45 1.25 1.21 0.65 0.68 0.61 0.60 14:0 (0.09) (0.05) (0.975)(0.03) (0.02) (0.04) (0.02) (0.02) (0.02) (0.00) LPA 1.09 1.06 1.41 1.430.64 0.67 0.67 0.65 0.75 0.77 20:4 (0.05) (0.03) (0.976) (0.01) (0.01)(0.05) (0.01) (0.04) (0.01) (0.01) CPA 1.19 1.19 1.45 1.60 1.05 0.970.88 0.83 0.71 0.76 16:0 (0.09) (0.01) (1.015) (0.03) (0.05) (0.04)(0.02) (0.04) (0.00) (0.01) LPA 1.18 1.10 1.47 1.55 0.96 0.79 0.87 0.850.97 1.03 18:1 (1.01) (0.13) (1.021) (0.02) (0.05) (0.06) (0.02) (0.03)(0.02) (0.00) CPA 1.06 1.05 1.56 1.47 0.79 0.79 0.85 0.82 0.84 0.89 18:0(0.03) (0.01) (1.097) (0.01) (1.01) (0.02) (0.02) (0.01) (0.01) (0.00)Total 5.83 5.73 7.33 7.50 4.69 4.44 3.91 3.63 3.88 4.06 CPA (0.22)(0.11) (5.102) (0.08) (0.08) (0.12) (0.03) (0.12) (0.04) (0.01)

TABLE 5C Difference between spiked and non-spiked Donor A Exp Avg/ DonorB Exp Avg/ Donor C Exp Avg/ Donor D Exp Avg/ Donor E Exp Avg/ Spiked LPAcone) Spiked LPA cone) Spiked LPA cone) Spiked LPA cone) Spiked LPAcone) HPLC HPLC HPLC HPLC HPLC Post- LC/ESI Post- LC/ESI Post- LC/ESIPost- LC/ESI Post- LC/ESI Column MS/MS Column MS/MS Column MS/MS ColumnMS/MS Column MS/MS CPA 0.41/ 0.41/ 0.47/ 0.41/ 0.49/ 0.53/ 0.41/ 0.46/0.43/ 0.42/ 14:0 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 LPA0.47/ 0.42/ 0.43/ 0.50/ 0.42/ 0.40/ 0.41/ 0.37/ 0.55/ 0.55/ 20:4 0.500.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 CPA 0.43/ 0.45/ 0.49/ 0.56/0.50/ 0.56/ 0.44/ 0.40/ 0.42/ 0.48/ 16:0 0.50 0.50 0.50 0.50 0.50 0.500.50 0.50 0.50 0.50 LPA 0.49/ 0.45/ 0.42/ 0.51/ 0.60/ 0.48/ 0.57/ 0.47/0.45/ 0.56/ 18:1 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0..50 0.50 CPA0.50/ 0.44/ 0.57/ 0.55/ 0.51/ 0.56/ 0.52/ 0.51/ 0.51/ 0.59/ 18:0 0.500.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 Total 2.31/ 2.17/ 2.37/2.53/ 2.52/ 2.53/ 2.35/ 2.20/ 2.36/ 2.60/ LPA 2.50 2.50 2.50 2.50 2.502.50 2.50 2.50 2.50 2.50 *Avg.: n = 3 σ = standard deviation

Example 4 GRBI Synthesis

The synthetic route for GRBI(1-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthene]-2-yl)guanidine)is described in Scheme 1. Compound 1 can be synthesized in 4 steps;starting from the commercially available Rhodamine B. Compound 4 wasobtained as described with a yield of 95%. The reaction of 4 with1,3-bis-Boc-2-methyl-2-thiopseudourea and HgCl₂ produced Boc-protectedcompound 5 in 90% yield. Guanidine deprotection was accomplished withTFA in DCM, which upon neutralization under basic condition gavecompound 1 in 60% yield.

2-amino-3′,6′-bis(diethylamino)spiro[isoindoline-1,9′-xanthen]-3-one (4)

To a stirred solution of 3 (1 g, 2.1 mmol) in ethanol (60 mL) at roomtemperature was added hydrazine monohydrate (98%, 0.6 mL, 12 mmol), andthe mixture was refluxed for 12 h. After cooling to room temperature,the solvent was evaporated under reduced pressure. The resulting solidwas dissolved in CHCl₃ (50 mL) and the organic phase was washed with H₂O(3×50 mL), followed by drying over anhydrous Na₂SO₄. After filtrationand removal of solvent, the crude product was purified by flashchromatography (DCM:MeOH=97.5:2.5) to afford a yellow oil product with ayield of 950 mg (95%). ¹H-NMR (600 MHz, CDCl₃) δ 1.16 (t, J=7.1 Hz,12H), 3.34 (q, J=7.1 Hz, 8H), 3.61 (s, 2H), 6.29 (dd, J=8.8 Hz, 2H),6.42 (d, J=2.5 Hz, 2H), 6.46 (d, J=8.8 Hz, 2H), 7.0 (m, 1H), 7.44 (m,2H), 7.93 (m, 1H). ¹³C-NMR (151 MHz, CDCl₃) δ 12.75, 44.51, 66.04,98.12, 104.74, 108.18, 123.13, 123.97, 128.23, 130.19, 132.63, 149.03,151.71, 153.99, 166.28. HR-ESI-MS (m/z) 457.2577[M+H]⁺.

(E)-tert-butyl(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthene]-2-ylamino)(tert-butoxy-carbonylamino)methylenecarbamate(5)

To a stirred solution of 4 (290 mg, 0.64 mmol), HgCl₂ (190 mg, 0.70mmol) and 1,3-bis-Boc-2-methyl-2-thiopseudourea (188 mg, 0.70 mmol) inanhydrous DMF (10 mL) under argon was added triethylamine (0.45 mL, 3.18mmol). The resulting suspension was stirred in an ice bath for 2 h, andthen at room temperature for 12 h. The mixture was diluted with CHCl₃and filtered through a short Celite column. The filtrate was washed withsaturated NaHCO₃ solution (25 mL) and H₂O (3×25 mL). The combinedorganic phase was dried over anhydrous Na₂SO₄. After filtration andremoval of solvent, the crude product was purified by flashchromatography (EtOAc:hexane=1:2) to afford a purple solid with a yieldof 410 mg (90%). ¹H-NMR (600 MHz, CDCl₃) δ 1.16 (t, J=6.9 Hz, 12H), 1.37(s, J=9.2 Hz, 9H), 1.38 (s, J=20.5 Hz, 9H), 3.33 (m, J=6.8 Hz, 8H), 6.27(dd, J=32.5, 8.2 Hz, 2H), 6.36 (d, J=14.9 Hz, 2H), 6.77 (s, 1H), 7.10(t, J=7.3 Hz, 1H), 7.46 (m, 2H), 7.94 (t, J=10.8 Hz, 1H), 9.33 (s, 1H),11.16 (s, 1H). ¹³C NMR (151 MHz, CDCl₃) δ 12.78, 28.07, 28.35, 44.47,66.68, 78.80, 83.25, 97.81, 104.55, 108.02, 123.60, 124.19, 128.02,128.23, 129.03, 133.21, 149.03, 152.35, 153.75, 156.39, 163.56.HR-ESI-MS (m/z) 699.3932 [M+H]⁺.

N-(9-(2-(2-carbamimidoylhydrazinecarbonyl)phenyl)-6-(diethylamino)-3H-xanthen-3-ylidene)-N-ethyl-ethanaminium2,2,2-trifluoroacetate (6)

To a stirred solution of 5 (380 mg, 0.55 mmol) in CH₂Cl₂ (2 mL) wasadded slowly a solution of trifluoroacetic acid (2 mL) in CH₂Cl₂ (2 mL).Stirring at room temperature was continued until the reaction wasadjudged complete by TLC analysis. Solvent and excess trifluoroaceticacid were evaporated under reduced pressure to afford thetrifluoroacetate salt as a dark purple solid for the next reactionwithout further purification.

1-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthene]-2-yl)guanidine(1)

To a stirred solution of 6 in anhydrous MeOH (3 mL), 0.5 M NaOMesolution (3 mL) was added, and the mixture was stirred at roomtemperature for 1 h. The solvent was evaporated under reduced pressure,and the resulting solid was dissolved in CHCl₃ (50 mL). The organicphase was washed with NaHCO₃ solution (25 mL) and H₂O (3×25 mL). Thecombined organic phase was dried over anhydrous Na₂SO₄. After filtrationand removal of solvent, the crude product was purified by flashchromatography (DCM:MeOH=9:1) to afford a purple solid with a yield of160 mg (60%). ¹H-NMR (600 MHz, DMSO) δ 1.08 (t, J=7.0 Hz, 3H), 3.30 (q,J=7.1 Hz, 8H), 5.51 (d, J=293.9 Hz, 4H), 6.27 (dd, J=8.9, 2.5 Hz, 2H),6.30 (d, J=2.5 Hz, 2H), 6.56 (d, J=7.3 Hz, 2H), 6.88 (d, J=6.9 Hz, 1H),7.42 (m, 2H), 7.71 (d, J=6.5 Hz, 1H). ¹³C-NMR (151 MHz, DMSO) δ 12.49,43.63, 65.29, 97.15, 105.99, 107.39, 121.78, 123.17, 127.77, 128.72,130.55, 131.59, 147.93, 152.34, 152.78, 158.53. HR-ESI-MS (m/z)499.2827[M+H]⁺.

Example 5 GRBII Synthesis

The synthetic route for GRBII(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthene]-2-carboximidamide)is described in Scheme 2. Compound 2 can be synthesized in 4 steps;starting from the commercially available Rhodamine B Base. Compound 8was obtained according to the procedure described below. Without furtherpurification, the reaction of 8 with1,3-Bis(tert-butoxycarbonyl)guanidine and K₂CO₃ produced Boc-protectedcompound 9 in 62% yield. Guanidine deprotection was accomplished withTFA in DCM, which upon neutralization under basic conditions gavecompound 2 in 52% yield.

N-(9-(2-(chlorocarbonyl)phenyl)-6-(diethylamino)-3H-xanthen-3-ylidene)-N-ethylethanaminium(8)

To a solution of 7 (500 mg, 1.13 mmol) in anhydrous 1,2-dichloroethane(5 mL), was added a solution of phosphorus oxychloride (0.26 mL, 2.8mmol) in 1,2-dichloroethane (5 mL) drop wise over 5 min, the mixture wasrefluxed for 4 h. After the reaction mixture was cooled to roomtemperature, the solvent was evaporated under reduced pressure to yieldrhodamine B acyl chloride without further purification.

E)-tert-butyl(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthene]-2-yl)methanediylidene-dicarbamate(9)

1,3-Bis(tert-butoxycarbonyl)guanidine (290 mg, 1.13 mmol) and K₂CO₃(0.62 g, 4.5 mmol) were dissolved in anhydrous acetonitrile (5 mL) underargon. The crude rhodamine B acyl chloride was dissolved in anhydrousacetonitrile (5 mL) and added drop wise to the solution over 3 h. After12 h, the solvent was evaporated under reduced pressure; the residue wasdissolved in chloroform and washed with saturated NaHCO₃ solution (25mL) and H₂O (3×25 mL), the organic phase was dried over anhydrousNa₂SO₄. After filtration and removal of solvent, the crude product waspurified by flash chromatography (EtOAc:Hexane=1:2) to afford a yellowsolid with a yield of 476 mg (62%). ¹H-NMR (600 MHz, CDCl₃) δ 1.13 (t,J=7.1 Hz, 12H), 1.35 (s, 9H), 1.41 (s, 9H), 3.30 (m, 8H), 6.18 (dd,J=8.8, 2.6 Hz, 2H), 6.30 (d, J=8.8 Hz, 2H), 6.35 (d, J=2.6 Hz, 2H), 7.16(d, J=7.7 Hz, 1H), 7.51 (m, 1H), 7.58 (td, J=7.5, 1.1 Hz, 1H), 7.94 (d,J=7.6 Hz, 1H), 10.75 (s, 1H). ¹³C-NMR (151 MHz, CDCl₃) δ 12.78, 28.15,28.18, 44.40, 68.30, 78.74, 81.78, 97.72, 106.68, 107.23, 123.59,125.09, 127.65, 128.75, 129.20, 135.16, 139.08, 148.97, 150.16, 153.42,154.20, 156.14, 171.05. HR-ESI-MS (m/z) 684.3794 [M+H]⁺.

N-(9-(2-(carbamimidoylcarbamoyl)phenyl)-6-(diethylamino)-3H-xanthen-3-ylidene)-N-ethylethanaminium2,2,2-trifluoroacetate (10)

To a stirred solution of 9 (300 mg, 0.44 mmol) in CH₂Cl₂ (2 mL) wasadded slowly a solution of trifluoroacetic acid (2 mL) in CH₂Cl₂ (2 mL).Stirring at room temperature was continued until the reaction wasdetermined to be complete by TLC analysis. Solvent and excess oftrifluoroacetic acid was evaporated under reduced pressure to afford thetrifluoroacetate salt as a dark purple solid for next reaction withoutfurther purification.

3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthene]-2-carboximidamide(2)

To a stirred solution of 10 in anhydrous MeOH (3 mL), 0.5 M NaOMesolution (3 mL) was added and the mixture was stirred at roomtemperature for 1 h. The solvent was evaporated under reduced pressureand the resulting solid was dissolved in CHCl₃ (50 mL), and the organicphase was washed with NaHCO₃ solution (25 mL) and H₂O (3×25 mL), thecombined organic phase was dried over anhydrous Na₂SO₄. After filtrationand removal of solvent, the crude product was purified by flashchromatography (DCM:MeOH=9:1) to afford a purple solid with a yield of110 mg (52%). ¹H-NMR (600 MHz, DMSO) δ 1.09 (t, J=7.0 Hz, 12H), 3.34 (q,J=7.1 Hz, 8H), 6.42 (dd, J=9.0, 2.6 Hz, 2H), 6.45 (d, J=2.5 Hz, 2H),6.72 (d, J=8.9 Hz, 2H), 7.01 (d, J=7.8 Hz, 1H), 7.58 (m, 1H), 7.68 (m,J=1.1 Hz, 1H), 7.69 (bs, 3H), 7.99 (d, J=7.7 Hz, 1H). ¹³C-NMR (151 MHz,DMSO) δ 12.38, 43.69, 79.18, 97.67, 103.08, 108.69, 123.65, 124.39,124.53, 127.22, 129.43, 136.51, 149.17, 151.72, 153.52, 153.64, 169.01.HR-ESI-MS (m/z) 484.2751 [M+H]⁺.

Example 6 LPA Universal Detection with GRBI and GRBII

The spiro guanidine rhodamines GRBI and GRBII were evaluated asuniversal fluorophores for the detection and quantification oflysophosphatidic acid (LPA).

LPA14:0, 16:0, 18:0, 18:1 were obtained by protonation of theircorresponding sodium salt respectively. 100 mg of LPA sodium salt weredissolved in 15 mL of a solvent mixture CHCl₃:MeOH (2:1), followed bythe addition of 2 mL DI water. After shaking the solution, the phaseswere allowed to separate, and the pH of the aqueous phase was adjustedwith 3 M HCl to pH 2.5-3.0. The organic phase was separated, and thesolvent removed under reduced pressure.

The fluorescence emission intensity of GRBI and GRBII in the presence of10 μM LPA16:0 in 2.5% DMSO/chloroform was evaluated. The final probeconcentration was 5 μM. An excitation wavelength of 550 nm was used, andfluorescence emission at 570 nm was measured. As shown in FIG. 9, GRBIIexhibited greater fluorescence emission in the presence of LPA 16:0 ascompared to GRBI.

Based on these results; the performance of the GRBII fluorophore for LPAdetection was further explored. From the evaluation of a series ofsolvent systems, as well as LPA sample preparation experiments, it wasfound that GRBII gave the same fluorescence enhancement in the presenceof the individual LPAs (LPA14:0, LPA16:0, LPA18:0 and LPA18:1) includedin this study. A DMSO titration in chloroform showed that a CHCl₃: DMSO9:1 mixture gave the best results (FIG. 10). In each case, the LPAconcentration was 10 μM, and the final GRBII concentration was 5 μM. Asseen in FIG. 9, GRBII produced the same fluorescence intensity with eachof the LPAs, demonstrating that GRBII can be used as a universal LPAdetection agent.

The fluorescence stability was monitored over time. As shown in FIG. 11,fluorescence intensity remained substantially constant over 30 minutes.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

We claim:
 1. A compound according to (a) general formula I

wherein: R¹-R⁶ independently are hydrogen, hydroxyl, thiol, C₁-C₁₀alkyl, carboxyalkyl, amino, C₁-C₁₀ alkoxy, or halogen, R⁷-R¹⁰independently are hydrogen, alkyl, acyl, carboxyl, nitro, amino,alkylamino, or —SO₃H, R¹¹ is N—C(═R¹³)—NH₂, N—C(NH₂)═N—C(═R¹³)—NH₂, orN—NH—C(NH₂)═N—C(═R¹³)—NH₂, where R¹³ is O, S, or NH, or R¹¹ isN—NH—C(═R¹³)—NH₂, where R¹³ is O or NH, each R¹² independently ishydrogen or lower alkyl, or each of R¹, R², R⁵, and R⁶ may together withan adjacent R¹² and N atom form a 6-membered heterocyclic ring, and X isO, S, CH₂, NH, or SiR¹⁴ where R¹⁴ is H or C₁-C₁₀ alkyl; or (b) generalformula II

wherein: R¹-R⁵, R¹⁵, R¹⁷, and R¹⁸ independently are hydrogen, hydroxyl,thiol, C₁-C₁₀ alkyl, carboxyalkyl, amino, C₁-C₁₀ alkoxy, or halogen, R¹⁶is —N(R¹²)₂, R⁷-R¹⁰ independently are hydrogen, alkyl, acyl, carboxyl,nitro, amino, alkylamino, or —SO₃H, R¹¹ is N—C(═R¹³)—NH₂,N—NH—C(═R¹³)—NH₂, N—C(NH₂)═N—C(═R¹³)—NH₂, or N—NH—C(NH₂)═N—C(═R¹³)—NH₂,where R¹³ is O, S, or NH, each R¹² independently is hydrogen or C₁-C₁₀alkyl, or each of R¹, R², R¹⁵, and R¹⁷ may together with an adjacent R¹²and N atom form a 6-membered heterocyclic ring, and X is O, S, CH₂, NH,or SiR¹⁴ where R¹⁴ is H or C₁-C₁₀ alkyl; or (c) general formula III

wherein: R²-R⁵, R¹⁵, R¹⁷, and R¹⁹-R²¹ independently are hydrogen,hydroxyl, thiol, C₁-C₁₀ alkyl, carboxyalkyl, amino, C₁-C₁₀ alkoxy, orhalogen, one of R¹⁶ and R¹⁸ is hydrogen, hydroxyl, thiol, lower alkyl,carboxyalkyl, amino, C₁-C₁₀ alkoxy, or halogen, and the other of R¹⁶ andR¹⁸ is —N(R¹²)₂, R⁷-R¹⁰ independently are hydrogen, alkyl, acyl,carboxyl, nitro, amino, alkylamino, or —SO₃H, R¹¹ is N—C(═R¹³)—NH₂,N—NH—C(═R¹³)—NH₂, N—C(NH₂)═N—C(═R¹³)—NH₂, or N—NH—C(NH₂)═N—C(═R¹³)—NH₂,where R¹³ is O, S, or NH, each R¹² independently is hydrogen or C₁-C₁₀alkyl, or if R¹⁶ is —N(R¹²)₂, each of R¹⁵, R¹⁷, R²⁰, and R²¹ maytogether with an adjacent R¹² and N form a 6-membered heterocyclic ring,and X is O, S, CH₂, NH, or SiR¹⁴ where R¹⁴ is H or C₁-C₁₀ alkyl; or (d)general formula IV

wherein: R¹-R⁴, R⁶, and R²²-R²⁴ independently are hydrogen, hydroxyl,thiol, C₁-C₁₀ alkyl, carboxyalkyl, amino, C₁-C₁₀ alkoxy, or halogen,R⁷-R¹⁰ independently are hydrogen, alkyl, acyl, carboxyl, nitro, amino,alkylamino, or —SO₃H, R¹¹ is N—C(═R¹³)—NH₂, N—NH—C(═R¹³)—NH₂,N—C(NH₂)═N—C(═R¹³)—NH₂, or N—NH—C(NH₂)═N—C(═R¹³)—NH₂, where R¹³ is O, S,or NH, each R¹² independently is hydrogen or C₁-C₁₀ alkyl, or each ofR¹, R², R²³, and R²⁴ may together with an adjacent R¹² and N form a6-membered heterocyclic ring, and X is O, S, CH₂, NH, or SiR¹⁴ where R¹⁴is H or C₁-C₁₀ alkyl.
 2. The compound of claim 1, wherein X is O.
 3. Thecompound of claim 1 according to: general formula I, wherein R¹-R¹⁰ areH; general formula II, wherein R¹-R⁵; R⁷-R¹⁰, R¹⁵, R¹⁷, and R¹⁸ arehydrogen; general formula III, wherein R²-R⁵; R⁷-R¹⁰, R¹⁵, R¹⁷, andR¹⁹-R²¹ are hydrogen, one of R¹⁶ and R¹⁸ is hydrogen, and the other ofR¹⁶ and R¹⁸ is —N(R¹²)₂; or general formula IV, wherein R¹-R⁴, R⁶⁻¹⁰,and R²²-R²⁴ are hydrogen.
 4. The compound of claim 1, wherein thecompound is


5. A method for quantifying lysophosphatidic acid species, comprising:combining a sample that may include one or more lysophosphatidic acidspecies with a compound according to claim 1 in a solvent comprisingdimethylsulfoxide in methanol to form a solution; exposing the solutionto a light source; measuring fluorescence intensity of the solution; anddetermining, based on the fluorescence intensity, a total concentrationof lysophosphatidic acid species in the sample.
 6. The method of claim5, further comprising obtaining the sample by extractinglysophosphatidic acid species from a sample comprising plasma or serum,wherein the sample comprising plasma or serum is obtained from a subjectsuspected of being at risk of a condition associated with an aberrantLPA level, the method further comprising determining a risk level forthe condition, wherein the risk level is based at least in part on thetotal concentration of lysophosphatidic acid species.
 7. The method ofclaim 5, wherein the compound is

and fluorescence intensity is measured at 570 nm.
 8. The method of claim6, wherein the condition is cancer, cardiovascular disease, plateletaggregation, ischemia perfusion injury, neuropathic pain, aneuropsychiatric disorder, a reproductive disorder, or fibrosis.
 9. Akit for detecting and quantifying lysophosphatidic acid, comprising atleast one compound according to claim
 1. 10. The kit of claim 9, whereinthe compound is


11. The method of claim 6, wherein the condition is ovarian cancer. 12.The method of claim 6, wherein extracting lysophosphatidic acid speciesfrom the sample comprising plasma or serum further comprises: combiningthe sample comprising plasma or serum with a an organic solventcomprising a C₁-C₁₀ alkyl alcohol and chloroform to form a mixture;separating organic and aqueous layers of the mixture; extracting theaqueous layer with a buffer at neutral pH to form an extracted aqueousphase; mixing the extracted aqueous phase with chloroform; separatingchloroform from the extracted aqueous phase to form a washed aqueousphase; adding phosphoric acid to the washed aqueous phase to form anacidified aqueous phase; loading the acidified aqueous phase onto asolid-phase extraction (SPE) cartridge including a stationary phasecomprising silica derivatized with hydrocarbon chains; flowing water andsubsequently chloroform through the SPE cartridge; drying the SPEcartridge; and flowing a C₁-C₁₀ alkyl alcohol through the SPE cartridge,thereby eluting lysophosphatidic acid species in the C₁-C₁₀ alkylalcohol from the SPE cartridge.
 13. The compound of claim 1, wherein R¹³is NH.
 14. The method of claim 12, wherein extracting lysophosphatidicacid species from the sample comprising plasma or serum furthercomprises: a chromatographic separation to measure levels of total LPAand individual LPA subspecies.
 15. The method of claim 14, wherein thechromatographic separation is performed using a reversed-phasehigh-performance liquid chromatography column.
 16. The method of claim5, wherein the solvent comprises dimethylsulfoxide in methanol.
 17. Themethod of claim 6, further comprising determining a risk level for thecondition, wherein the risk level is based at least in part on the totalconcentration of lysophosphatidic acid species.
 18. The method of claim12, wherein the organic solvent comprises a C₁-C₁₀ alkyl alcohol andchloroform.